Project Summary Adult stem cells supporting different tissues can meet the dual demands of lifelong persistence and continued production of daughter cells that differentiate through a variety of strategies, which have presumably evolved to suit demand. Highly proliferative stem cells that support continuously renewing epithelia in skin and intestines are especially important targets of investigation to understand how a large replicative burden is distributed, how mutations initiate pre-cancerous amplification, and how stem cells might be used for regenerative therapies. Somatic follicle stem cells (FSCs) in the Drosophila ovary provide an exceptional opportunity to examine highly proliferative epithelial stem cell organization, dynamics and regulation in detail. This proposal builds on extensive prior use of genetic methods for lineage tracing and direct imaging of stem cells and their progeny to construct a sophisticated and much revised picture of the location, dynamics and immediate products of a community of FSCs in their niche. The arrangement of FSCs has remarkable similarities to mammalian intestinal stem cells. In both cases, a group of about sixteen stem cells is maintained by a population asymmetry mechanism where stem cell division and differentiation are not coupled. Importantly, this leads inevitably to stabilization and amplification of faster-proliferating mutant stem cell lineages. Both types of stem cell also exhibit heterogeneous behaviors along the major developmental axis but individual stem cells also can exchange positions (?dynamic heterogeneity?). FSCs in different locations directly produce two different cell types. For FSCs, genetic interrogation of signaling pathways has defined three principal, graded external signals that regulate FSC behavior. These insights will be pursued further to build an integrated picture of FSC behavior and how it is regulated by niche signals. One objective is to define how spatially graded FSC proliferation is imposed by graded JAK-STAT and Wnt pathway signaling. A second objective is to define how these same signals specify the location and alternative differentiation outcomes for individual FSCs and the community as a whole. A third objective is to understand how FSCs and associated niche cells develop prior to adulthood under the guidance of emerging patterns of external signals. The insights gained will provide information about how stem cells co-operate and compete within a community, how niche cells communicate with stem cells, a better understanding of how some cancers can initiate and how stem cell behavior might be manipulated for therapeutic benefit.